We are interested in the structure and function of enzyme systems that glycosylate proteins in the endoplasmic reticulum (ER) and that process glycoprotein carbohydrate units in the Golgi. ER systems under study include dolichol phosphate mannose synthase and the Saccharomyces ALG proteins which catalyze synthesis of the ER dolichol linked oligosaccharide. This oligosaccharide is transferred to specific asparagine residues of nascent polypeptide chains as they enter the ER lumen. Three ALG genes have recently been cloned and sequenced. We are especially interested in the structural and functional interaction among these enzymes. We will apply techniques such as the "synthetic lethal" approach to isolate genes for proteins involved in these interactions. Golgi glycoprotein processing systems under study include a family of Saccharomyces mannosyl transferases and the GDPase which plays a role in the transport of nucleotide sugars into the Golgi lumen. The transferases will be characterized biochemically, and mutations in genes which require the GDPase for maintenance of cell viability will be isolated by the synthetic lethal method. We will also explore a newly discovered mnn9 gene family which encodes proteins which may be required for a number of important Golgi functions. Since several of these systems are clearly "non-mammalian", they will also be studied as potential drug targets in the pathogenic yeast Candida albicans. We already have data on the Candida mannosyl transferases and have gene fragments from Candida mnn9 homologs. These genes will be "knocked out" and resulting strains will be tested for pathogenicity in a mouse model. Other Candida studies are aimed at characterizing genes and enzymes involved in the synthesis of the cell wall beta-mannosyl residues that may play a role in pathogenicity of the organism.